Galleria mellonella (Lepidoptera: Pyralidae) Hemocytes Release Extracellular Traps That Confer Protection Against Bacterial Infection in the Hemocoel.

Extracellular traps (ETs) released from vertebrate and invertebrate immune cells consist of chromatin and toxic granule contents that are capable of immobilizing and killing microbes. This recently described innate immune response is not well documented in insects. The present study found that ETs were released by hemocytes of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) in vivo and ex vivo after bacterial stimulation. ET release (ETosis), hemolymph coagulation, and melanization likely contributed to the immobilization and killing of the bacteria. The injection of G. mellonella hemocyte deoxyribonucleic acid (DNA) in the presence of bacteria increased bacterial clearance rate and prolonged insect survival. Taken together, these results indicate the presence of insect hemocyte extracellular traps (IHETs) that protect the insect against microbial infection in the hemocoel and represent the first documentation of ETs in insects in vivo.

The Galleria mellonella-Enteropathogenic Escherichia coli Model System: Characterization of Pathogen Virulence and Insect Immune Responses.

The use of Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae), an economical insect model, for the study of enteropathogenic Escherichia coli (Migula) (EPEC), a diarrheagenic human pathogen, has been demonstrated previously but remains poorly understood. The present study characterizes the Galleria-EPEC system extensively for future studies using this system. We found that EPEC causes disease in G. mellonella larvae when injected intrahemocoelically but not orally. Disease manifests as increased mortality, decreased survival time, delayed pupation, decreased pupal mass, increased pupal duration, and hemocytopenia. Disease symptoms are dose-dependent and can be used as metrics for measuring EPEC virulence in future studies. The type III secretion system was only partially responsible for EPEC virulence in G. mellonella while the majority of the virulence remains unknown in origin. EPEC elicits insect anti-bacterial immune responses including melanization, hemolymph coagulation, nodulation, and phagocytosis. The immune responses were unable to control EPEC replication in the early stage of infection (≤3 h post-injection). EPEC clearance from the hemocoel does not guarantee insect survival. Overall, this study provided insights into EPEC virulence and pathogenesis in G. mellonella and identified areas of future research using this system.

Honey bees with a drinking problem: potential routes of Nosema ceranae spore transmission.

Nosema apis and N. ceranae are the two causative agents of Nosema disease in adult honey bees (Apis mellifera L.). Nosema apis has been a recognized parasite for over a century and its epizootiology is well known. In contrast, N. ceranae is an emerging parasite of honey bees, which is now globally prevalent and the dominant Nosema spp. in many parts of the world. Despite this, many gaps in our knowledge exist regarding this species. For example, we do not fully understand all of the routes of transmission of N. ceranae among bees, or how long this parasite is capable of surviving in honey bee colonies. Here we investigated the viability and infectivity of N. ceranae spores in water and 2 M sucrose over time after storage at 33, 20, −12 and −20°C. Spores in both 2 M sucrose and water maintained high viability, except in water at −20°C over the course of the 6-week experiment. Infectivity was variable for spores after storage at all four temperatures, but all were infective at the last time point. The results provide evidence for cold tolerance and suggest that both water and 2 M sucrose (fall bee feed) could act as routes of transmission for N. ceranae. This work also contains information that may help influence management recommendations for the parasite.

Nosema ceranae (Microspora: Nosematidae): A Sweet Surprise? Investigating the Viability and Infectivity of N. ceranae Spores Maintained in Honey and on Beeswax.

Nosema disease is a prominent malady among adult honey bees [Apis mellifera L. (Hymenoptera: Apidae)], caused by the microsporidian parasites, Nosema apis Zander (Microspora: Nosematidae) and N. ceranae Fries et al. 1996. The biology of N. apis is well understood, as this parasite was first described over a century ago. As N. ceranae is an emerging parasite of the honey bee, we do not yet understand how long spores of this parasite survive in honey bee colonies, or all the potential modes of transmission among bees. We investigated the viability and infectivity of N. ceranae spores in honey and on beeswax over time after exposure to 33, 20, -12, and -20°C. Spores in honey maintained viability at freezing temperatures for up to 1 yr and remained viable considerably longer than those on beeswax. Based on this evidence, honey may act as an important reservoir for infective spores to initiate or perpetuate N. ceranae infections in honey bee colonies. This work provides information that may help enhance current management recommendations for apiculturalists.

Disruption of redox homeostasis leads to oxidative DNA damage in spermatocytes of Wolbachia-infected Drosophila simulans.

Molecular interactions between symbiotic bacteria and their animal hosts are, as yet, poorly understood. The most widespread bacterial endosymbiont, Wolbachia pipientis, occurs in high density in testes of infected Drosophila simulans and causes cytoplasmic incompatibility (CI), a form of male-derived zygotic lethality. Wolbachia grow and divide within host vacuoles that generate reactive oxygen species (ROS), which in turn stimulate the up-regulation of antioxidant enzymes. These enzymes appear to protect the host from ROS-mediated damage, as there is no obvious fitness cost to Drosophila carrying Wolbachia infections. We have now determined that DNA from Wolbachia-infected mosquito Aedes albopictus (Aa23) cells shows a higher amount of the base 8-oxo-deoxyguanosine, a marker of oxidative DNA damage, than DNA from uninfected cells, and that Wolbachia infection in D. simulans is associated with an increase in DNA strand breaks in meiotic spermatocytes. Feeding exogenous antioxidants to male and female D. simulans dramatically increased Wolbachia numbers with no obvious effects on host fitness. These results suggest that ROS-induced DNA damage in sperm nuclei may contribute to the modification characteristic of CI expression in Wolbachia-infected males and that Wolbachia density is sensitive to redox balance in these flies.

Evaluation of lysozyme-HCl for the treatment of chalkbrood disease in honey bee colonies.

Chalkbrood, caused by Ascosphaera apis (Maassen and Claussen) Olive and Spiltor, is a cosmopolitan fungal disease of honey bee larvae (Apis mellifera L.) for which there is no chemotherapeutic control. We evaluated the efficacy of lysozyme-HCl, an inexpensive food-grade antimicrobial extracted from hen egg white, for the treatment of chalkbrood disease in honey bee colonies. Our study compared three doses of lysozyme-HCl in sugar syrup (600, 3,000, and 6,000 mg) administered weekly for 3 wk among chalkbrood-inoculated colonies, colonies that were inoculated but remained untreated, and colonies neither inoculated or treated. Lysozyme-HCl at the highest dose evaluated was found to suppress development of chalkbrood disease in inoculated colonies to levels observed in uninoculated, untreated colonies, and did not adversely affect adult bee survival or brood production. Honey production was significantly negatively correlated with increased disease severity but there were no significant differences in winter survival among treatment groups. Based on our results, lysozyme-HCl appears to be a promising, safe therapeutic agent for the control of chalkbrood in honey bee colonies.

Plant nutrients and the spatiotemporal distribution dynamics of Ceutorhynchus obstrictus (Coleoptera: Curculionidae) and its parasitoids.

The cabbage seedpod weevil, Ceutorhynchus obstrictus (Marsham), is an alien invasive pest of canola, Brassica napus L., in North America. Field populations of both adults and larvae are known to be aggregated in their distributions, but the causal mechanism for this clustering is not understood. We investigated the hypothesis that spatial distributions of C. obstrictus adults, larvae, and their parasitoids were directly or indirectly related to host plant quality as indicated by leaf tissue nutrient contents. During the years of 2005 and 2007, these insect populations were sampled in a grid pattern in three commercial fields of B. napus located near Lethbridge, Alberta, Canada. Leaf samples were collected from plants in each grid plot and analyzed for nutrient contents. Spatial patterns and associations among the collected variables were analyzed using Spatial Analysis by Distance IndicEs software. We observed significant spatial associations among C. obstrictus adults, larvae, and parasitoids. The distributions of C. obstrictus adults and larvae were correlated with several plant nutrients, most notably nitrogen and sulfur. Evidence was found for a possible nitrogen-sulfur interaction in which ovipositioning C. obstrictus females appeared to prefer plants with high levels of sulfur and low levels of nitrogen. Spatial tracking of C. obstrictus larvae by its parasitoids was evident in only one field, and this appeared related to comparatively low parasitoid populations in two of the three fields. The associations between C. obstrictus and plant nutrients may have possible applications in fertility management, trap cropping, and precision insecticide application technology.

The endosymbiont Wolbachia pipientis induces the expression of host antioxidant proteins in an Aedes albopictus cell line.

Wolbachia are obligate intracellular bacteria which commonly infect arthropods. They are maternally inherited and capable of altering host development, sex determination, and reproduction. Reproductive manipulations include feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility. The mechanism by which Wolbachia avoid destruction by the host immune response is unknown. Generation of antimicrobial peptides (AMPs) and reactive oxygen species (ROS) by the host are among the first lines of traditional antimicrobial defense. Previous work shows no link between a Wolbachia infection and the induction of AMPs. Here we compare the expression of protein in a cell line naturally infected with Wolbachia and an identical cell line cured of the infection through the use of antibiotics. Protein extracts of each cell line were analyzed by two dimensional gel electrophoresis and LC/MS/MS. Our results show the upregulation of host antioxidant proteins, which are active against ROS generated by aerobic cell metabolism and during an immune response. Furthermore, flow cytometric and microscopic analysis demonstrates that ROS production is significantly greater in Wolbachia-infected mosquito cells and is associated with endosymbiont-containing vacuoles located in the host cell cytoplasm. This is the first empirical data supporting an association between Wolbachia and the insect antioxidant system.

Molecular and functional characterization of granulin-like molecules of insects.

Granulins are a group of highly conserved growth factors that have been described from a variety of organisms spanning the metazoa. Here, we report on the identification of two partial transcripts encoding granulin-like molecules from Aa23 embryonic cells of Aedes albopictus and primary haemocytes from Manduca sexta. Both these partial transcripts had the characteristic 12-cysteine motif that is a hallmark of the granulin family and they represent the first granulin mRNA transcripts identified from insects. Moreover, we demonstrate that the recombinant granulin molecule that we originally cloned in the goldfish, induced proliferation of both Aa23 embryonic cells and primary haemocytes. Interestingly, this proliferative effect was upregulated in the presence of the intracellular symbiotic bacterium Wolbachia pipientis. Thus, granulin appears to be a highly conserved growth factor not only in lower vertebrates but also invertebrates.

Differences in Phyllotreta cruciferae and Phyllotreta striolata (Coleoptera: Chrysomelidae) responses to neonicotinoid seed treatments.

Insecticidal seed treatments are used commonly throughout the Northern Great Plains of North America to systemically protect seedlings of canola (Brassica napus L. and Brassica rapa L.) from attack by the flea beetles Phyllotreta cruciferae (Goeze) and Phyllotreta striolata (F.) (Coleoptera: Chrysomelidae). Here, we investigated differential responses by the two flea beetle species to the neonicotinoid seed treatments thiamethoxam (Helix and Helix XTra) and clothianidin (Prosper 400) in greenhouse experiments. P. cruciferae experienced higher mortality and fed less when exposed to these compounds than did P. striolata. Beetles of the overwintered and the summer generations responded differently when feeding on seedlings that developed with insecticidal seed treatments, with mortality higher for P. cruciferae in May than in August. When the two flea beetle species were held together at equal densities and allowed to feed on seedlings affected by the seed treatments, mortality of P. cruciferae significantly exceeded that of P. striolata. Differences in efficacies of these compounds for these beetles have ramifications for management strategies in regions where these insects occur sympatrically. Competitive release of P. striolata was previously reported to occur when P. cruciferae was excluded from brassicaceous crops; consequently, the consistent use of these seed treatments over millions of hectares of canola cropland may be a factor that contributes to a shift in prevalence of flea beetle pest species from P. cruciferae toward P. striolata.

Resistance of some cultivated Brassicaceae to infestations by Plutella xylostella (Lepidoptera: Plutellidae).

Selecting insect-resistant plant varieties is a key component of integrated management programs of oligophagous pests such as diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), but rigorous research on important life history parameters of P. xylostella in relation to host plant resistance is rare. We evaluated six conventional brassicaceous species, namely, Brassica napus L. 'Q2', B. rapa L., B. juncea (L.) Czern., B. carinata L., B. oleracea L., and Sinapis alba L., and two herbicide-tolerant cultivars, namely, B. napus 'Liberty' and B. napus 'Conquest' for their resistance against P. xylostella. Brassicaceae species and cultivars varied considerably in their susceptibilities as hosts for P. xylostella. Sinapis alba and B. rapa plants were highly preferred by ovipositing females and trichome density on adaxial and abaxial leaf surfaces had nonsignificant effects on P. xylostella oviposition. Larval survival was similar on the genotypes we tested, but host plants significantly affected larval and pupal developmental time, herbivory, pupal weight, silk weight, adult body weight, forewing area and longevity (without food) of both male and female P. xylostella. Larval and pupal development of females was fastest on B. juncea and S. alba, respectively. Specimens reared on B. napus Liberty and B. oleracea, respectively, produced the lightest female and male pupae. Defoliation by both female and male larvae was highest on B. rapa, whereas least herbivory occurred on S. alba. Females reared on S. alba were heavier and lived longer in the absence of food than their counterparts raised on other tested host plants. Brassica oleracea could not compensate for larval feeding to the level of the other species we evaluated. B. napus Conquest, B. napus Q2, B. carinata, B. rapa, and S. alba produced, respectively, 1.6-, 1.8-, 1.8-, 3.9-, and 5.5-fold heavier root systems when infested than their uninfested counterparts, suggesting that these species were better able to tolerate P. xylostella infestations.

Isolation and characterization of plaque-purified strains of Malacosoma disstria Nucleopolyhedrovirus.

Seven plaque-purified genotypic variants or strains, derived from a previously described field isolate of the Malacosoma disstria Nucleopolyhedrovirus (MadiNPV) from Alberta populations of forest tent caterpillar, were characterized based on distinctive restriction endonuclease fragment patterns. Two strains, MadiNPV-pp3 and MadiNPV-pp11, were selected for further characterization, as they represented strains producing high and low budded virus (BV) titres, respectively, in the M. disstria cell line UA-Md203. Analysis of restriction endonuclease fragment profiles indicated the genomes differed significantly in size, 133.8 +/- 2.4 kb for MadiNPV-pp3 and 118.1 +/- 3.5 kb for MadiNPV-pp11. These strains were characterized based on their BV production in three different cell lines derived from M. disstria haemocytes. Compared with MadiNPV-pp11, MadiNPV-pp3 produced two- to three-fold more BVs in UA-Md203 and 210 other cell lines; however, BV production was only marginally higher for MadiNPV-pp3 in the UA-Md221 cell line. Similarly, the yield of polyhedral inclusion bodies was significantly higher for MadiNPV-pp3 in UA-Md203 and 210 cell lines than for MadiNPV-pp11 but not in the UA-Md221 cell line. This data, although derived from a limited number of cell lines, suggested MadiNPV-pp3 may have a broader tissue tropism than MadiNPV-pp11.

Identification and genomic analysis of a second species of nucleopolyhedrovirus isolated from Mamestra configurata.

MacoNPV-96B is a nucleopolyhedrovirus isolated from naturally infected Mamestra configurata (Lepidoptera: Noctuidae) larvae. It was initially identified due to its completely different restriction endonuclease profile relative to the previously sequenced Mamestra configurata virus MacoNPV-90/2 (Q. Li, C. Donly, L. Li, L. G. Willis, D. A. Theilmann, and M. Erlandson, 2002, Virology 294, 106-121). The MacoNPV-96B host range and virulence were also found to differ significantly from those of the previous isolate. To further understand the complex of viruses infecting M. configurata, the genome of MacoNPV-96B was completely sequenced and analyzed in comparison with the genome of MacoNPV-90/2 and other sequenced baculoviruses. MacoNPV-96B consists of 158,482 bp, and 168 open reading frames (ORFs) of 150 nucleotides or longer with minimal overlap have been identified. The genome of MacoNPV-96B is 3422 bp larger than MacoNPV-90/2 and although gene arrangement is virtually identical, there are 9 ORFs unique to MacoNPV-96B and 10 unique to MacoNPV-90/2. bro genes were found to be associated with nonhomologous regions, suggesting that bro genes may facilitate recombination between genomes. A major difference in the gene content between the two viruses is a 5.4-kb insert in MacoNPV-96B, which is highly homologous to a cluster of Xestia c-nigrum granulovirus (XecnGV) ORFs, suggesting recent recombination events between these two viruses. Nucleotide sequence and amino acid sequence identity between the common ORFs of MacoNPV-96B and MacoNPV-90/2 average 87 and 90%, respectively. The sequence data suggest that MacoNPV-96B and MacoNPV-90/2 are closely related but have diverged and evolved into two separate species. This is the first study to identify highly related but separately evolving viruses in the same insect host and geographic location. A new Identity-GeneParityPlot analysis was developed to perform a comparison of two viral genomes in gene content and arrangement as well as homology level of individual ORFs.

Isolation and characterization of a Chryseobacterium strain from the gut of the American cockroach, Periplaneta americana.

A 16S rDNA sequence cloned directly from whole-gut microbiota of the American cockroach, Periplaneta americana, indicated the presence of a member of the Bacteroides/Flavobacterium group most closely related to the genus Flavobacterium. In an attempt to confirm this finding, we isolated a yellow-pigmented bacterium (strain FR2) from the hindgut of this insect. Strain FR2 was phylogentically and phenotypically most similar to species of Flavobacterium and related bacteria, namely Chryseobacterium indologenes. Fifty-four other yellow-pigmented bacteria isolated during a 1-year study shared the salient phenotypic characteristics of Chryseobacterium spp., and thus were considered the same phenotype. This phenotype's abundance was related to the fiber content of the insect diet, being consistently detected only in cockroaches fed a high-fiber diet (30% crude fiber by weight). The highest population density was in the hindgut, ranging from 2 x 10(6) to 1.2 x 10(7) colony forming units ml(-1) during a 1-year period. The nature of the symbiosis between the FR2 phenotype and P. americana is discussed.

Disease prevalence and transmission of Microsporidium phytoseiuli infecting the predatory mite, Phytoseiulus persimilis (Acari: Phytoseiidae).

Isolated colonies of the predatory mite, Phytoseiulus persimilis, were used to gain information regarding prevalence and transmission of Microsporidium phytoseiuli. Two colonies of P. persimilis were reared on spider mite (Tetranychus urticae)-infested bean plants in isolated cages. Disease prevalence of predators from Colony 1 remained relatively low (between 0 and 15%) over 57 weeks of observation whereas disease prevalence of predators from Colony 2 increased over 3 months (from 12 to 100%). Disease prevalence among predators from Colony 1 had increased to 100% 2 months after weekly sampling had ceased for this colony and periodic sampling confirmed that disease prevalence among individuals of both colonies remained at 100%. Microsporidian spores were not detected in randomly chosen samples of T. urticae prey mites that were removed and examined biweekly during this period. Although numerous microsporidian spores were observed in smear preparations of fecal pellets examined by light microscopy, spores were not observed on leaf surfaces or predator feces when examined by SEM. The latter appeared as intact aggregates composed of numerous dumbbell-shaped crystals and it is unlikely that spores are liberated from intact fecal pellets onto leaf surfaces. Vertical transmission of M. phytoseiuli was 100%; horizontal transmission was low (14.3%) and occurred only when immature P. persimilis were permitted to develop in contact with infected immature and adult predators. The mean number of eggs produced per mated pair was highest when uninfected females were mated with uninfected males (63.2 eggs per mated pair). Although mean egg production decreased when one or both parents were infected, not all differences were significant. Male predatory mites did not contribute to infection of their progeny. Results suggest that routine examination of P. persimilis for microsporidian spores is essential for the management of M. phytoseiuli within P. persimilis colonies. Low disease prevalence and lack of obvious disease signs or symptoms, as in the case of M. phytoseiuli, increase the probability that these pathogens will escape notice unless individuals are routinely examined for pathogens.

Development and pathology of two undescribed species of microsporidia infecting the predatory mite, Phytoseiulus persimilis Athias-Henriot.

Two undescribed species of microsporidia were found in mass-reared Phytoseiulus persimilis Athias-Henriot from two commercial sources during a routine examination of these predators for pathogens. Both microsporidian species were described from specimens that had been prepared for transmission electron microscopy; live specimens were unavailable for examination. One microsporidium, identified as Species A, was described from two specimens obtained from a commercial insectary in North America. All observed stages of this microsporidium were uninucleate. Rounded-to-ovoid schizonts appeared to develop in direct contact with the cytoplasm of lyrate organ cells (ovarian tissue). Mature spores of Species A were elongate-ovoid and measured 2.88 x 1.21 microm. A polar filament coiled 7 to 10 times in the posterior half of the spore. Sporoblasts and spores were observed in the cytoplasm of cells of numerous tissues and in developing eggs within gravid females. A second species, identified as Species B, was described from five specimens obtained from a commercial source in Israel. All observed stages of this microsporidium were uninucleate. Schizonts of Species B were observed within the cytoplasm of cecal wall cells and within the nuclei of lyrate organ cells. Mature spores were ovoid and measured 2.65 x 1.21 microm. A polar filament coiled 3 to 4 times in the posterior half of the spore. Densely packed ribosomes often concealed the polar filament and other internal spore characteristics. Spores were observed in the cytoplasm of cells of numerous tissues and occasionally within the nuclei of lyrate organ cells. Numerous spores and presporal stages were observed within the ovary and developing eggs. The development and pathology of Species A and B were compared to those of Microsporidium phytoseiuli Bjøornson, Steiner and Keddie, a microsporidium previously described from P. persimilis obtained from a commercial source in Europe. The occurrence of three species of microsporidia within P. persimilis from three sources raises questions regarding the origin of these pathogens. Because microsporidia may have profound impact on the performance of P. persimilis, consideration must be given to the identification and exclusion of microsporidia from field-collected specimens or from predators that may be shared among commercial sources.

Significance of methanogenic symbionts for development of the American cockroach, Periplaneta americana.

The effects of suppression of methanogenesis with a drug, 2-bromoethanesulfonic acid (BES), on the hindgut ecosystem and development of the cockroach Periplaneta americana fed either low or high fiber diet were evaluated by measuring methane production, volatile fatty acids (VFA) concentrations in the hindgut, cockroach weight gain and development time (the length of nymphal period). Methane production and VFA concentrations in the hindgut of cockroaches fed high fiber diet were significantly higher than those fed low fiber diet. Although BES treatment greatly reduced methane production, VFA concentrations in the hindgut, cockroach weight gain and development time were not significantly altered. These results indicate that methanogenic microbes are not essential for keeping low hydrogen pressure in the hindgut lumen, and normal cockroach development.

A new tissue technique for evaluating effects of Bacillus thuringiensis toxins on insect midgut epithelium.

Epithelial tissue wholemounts were produced after enzymatic removal of basal lamina and connective tissue from midguts of Trichoplusia ni larvae. Wholemounts were nourished in artificial hemolymph and tissue viability was assessed for up to 24 hr using the vital dyes trypan blue, acridine orange (AO), propidium iodide (PI), and 4', 6-diamidino-2-phenylindole (DAPI). Peritrophic membrane synthesis and modification of Bacillus thuringiensis Cry1Ac protoxin to active toxin confirmed some normal epithelial function. Vital staining using the combination of AO and PI, or DAPI revealed altered membrane permeability in columnar epithelial and regenerative cells of tissues treated with activated Cry1Ac toxin while feeding and oral inoculation bioassays verified Cry1Ac toxicity. DAPI was selected to identify target cells in a rapid and highly sensitive assay.

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni.

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.

The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host.

An immunohistochemical study was conducted to detect the temporal infection sequence of Autographa californica M nuclear polyhedrosis virus in Trichoplusia ni larvae. Staining patterns indicated that the initial infection occurred in the midgut, simultaneously in columnar epithelial and regenerative cells, but that subsequently this tissue recovered. A major envelope glycoprotein stained in a polar fashion when it was expressed in columnar epithelial cells, but not when expressed in other cells types. Systemic infection was mediated by free virus for some tissues whereas infected hemocytes appeared to spread virus to other tissues by an unknown mechanism. A cell to cell spread within several tissues was detected. These results have important implications for baculoviruses engineered for improving their pesticide potential.